Methods and device for analyte characterization

ABSTRACT

The methods and apparatus, disclosed herein are of use for sequencing and/or identifying proteins, polypeptides and/or peptides. Proteins containing labeled amino acid residues may be synthesized and passed through nanopores. A detector operably coupled to a nanopore may detect labeled amino acid residues as they pass through the nanopore. Distance maps for each type of labeled amino acid residue may be compiled. The distance maps may be used to sequence and/or identify the protein. Apparatus of use for protein sequencing and/or identification is also disclosed herein. In alternative methods, other types of analytes may be analyzed by the same techniques.

RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 10/138,157, filed on May 1, 2002.

BACKGROUND

1. Field

The disclosed methods and apparatus relate to the analysis of analytesincluding, but not limited to, proteins, polypeptides, peptides, lipidsand polysaccharides. In particular, the methods and apparatus relates toprotein, polypeptide and/or peptide identification and/or sequencing.

2. Related Art

Identification and/or sequencing of analytes, such as proteins, arecritical for medical diagnostics, forensics, toxicology, pathology,biological warfare, public health and numerous other fields. The abilityto identify a particular pathogen or agent may depend on identificationof one or more specific analytes characteristic of that pathogen oragent. Identification of regulatory pathways involved in diseaseprocesses, metabolism, growth and cell division may depend onidentification and/or sequencing of analytes. Although a great deal ofresearch is presently directed towards identification and/or sequencingof nucleic acids or proteins, other analytes such as carbohydrates,polysaccharides, lipids, fatty acids, etc. may be of importance. Themethods and apparatus disclosed herein are focused on identificationand/or sequencing of proteins, polypeptides and peptides. However, theyare also of use for analysis of other types of analytes.

Existing methods for protein sequencing, based on the Edman degradationtechnique, are limited by the length of the protein that can besequenced. Accurate sequence determination is limited to about 50 to 100amino acid residues per sequencing run. Sequencing of longer proteins,which may be thousands of amino acid residues in length, requirescleavage into smaller fragments and assembly of overlapping shortsequences. The process is laborious, expensive, inefficient andtime-consuming and typically requires the use of radioactive labels andother hazardous chemicals, which can pose safety and waste disposalproblems.

A variety of techniques are available for identification of proteins,polypeptides and peptides. Commonly, these involve binding and detectionof antibodies that can recognize one or more epitopic domains on theprotein. Although antibody-based identification of proteins is fairlyrapid, such assays may occasionally show unacceptably high levels offalse positive or false negative results, due to cross-reactivity of theantibody with different antigens, low antigenicity of the target analyte(leading to low sensitivity of the assay), non-specific binding ofantibody to various surfaces, etc. They also require the preparation ofantibodies that can recognize an individual protein or peptide. As such,they are not suitable for the identification of novel proteins that havenot previously been characterized. More recently, mass spectroscopy hasbeen used for peptide identification and/or sequencing. Proteins andpolypeptides may be cleaved into smaller fragments and the amino acidcomposition of the fragments may be identified by mass spectroscopy.Analysis of a sufficient number of overlapping fragments can providedata on amino acid sequence. This process is also laborious, expensiveand requires substantial purification of the protein or peptide to beanalyzed.

A need exists in the art for methods and apparatus suitable for theidentification and/or sequencing of analytes, including proteins andpeptides that have not previously been identified or characterized.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain aspects of the disclosed methods andapparatus. The methods and apparatus may be better understood byreference to one or more of these drawings in combination with thedetailed description presented herein.

FIG. 1 is a flow chart illustrating a non-limiting exemplary apparatus100 (not to scale) and methods for protein sequencing 150 and/oridentification 160 by generation of distance maps 140.

FIG. 2 illustrates a non-limiting example of a sub-device 200 (not toscale) for protein 230 sequencing and/or identification byphotodetection.

FIG. 3 illustrates another non-limiting example of a sub-device 300 (notto scale) for protein 310 sequencing and/or identification by electricaldetection.

FIG. 4 shows non-limiting examples of protein labeling on cysteineresidues.

FIG. 5 shows non-limiting examples of protein labeling on lysine,arginine and N-terminal residues.

FIG. 6 shows non-limiting examples of protein labeling on aspartate,glutamate and C-terminal residues.

FIG. 7 shows non-limiting examples of protein labeling on serine andthreonine residues.

FIG. 8 shows the Raman spectra of four different types of nucleotides.Characteristic Raman emission peaks for each different type ofnucleotide. The data were collected without surface-enhancement orlabeling of the nucleotides.

FIG. 9 shows a comparative SERS spectrum of a 500 nM solution ofdeoxyadenosine triphosphate covalently labeled with fluorescein (uppertrace) and unlabeled dATP (lower trace). The dATP-fluorescein wasobtained from Roche Applied Science (Indianapolis, Ind.). A strongincrease in the SERS signal was detected for the fluorescein labeleddATP.

FIG. 10 illustrates the Raman spectrum of 1 mM tryptophan, taken with a1 second (upper trace) and 0.1 second (lower trace) collection time.

FIG. 11 shows the Raman spectrum of 1 mM cysteine, with a 0.1 secondcollection time.

FIG. 12 exemplifies the Raman spectrum of 1 mM methionine, with a 1second collection time.

FIG. 13 illustrates the Raman spectrum of 1 mM histidine, with a 1second collection time.

FIG. 14 shows the Raman spectrum of 1 mM phenylalanine, with a 1 secondcollection time.

FIG. 15 shows the Raman spectrum of 1 mM arginine, with a 0.1 secondcollection time.

FIG. 16 shows the Raman spectrum of 1 mM tyrosine, with a 1 secondcollection time (upper trace) and 0.1 second collection time (lowertrace).

FIG. 17 shows the Raman spectra of 1 mM 5-fluorotryptophan, with a 0.1second collection time.

FIG. 18 illustrates the Raman spectrum of 1% fetal calf serum, dried onan aluminum plate, with a 1 second collection time.

FIG. 19 shows the Raman spectrum of 100% whole calf serum, with a 1second collection time.

FIG. 20 shows the Raman spectrum of 0.1% whole calf serum, with a 1second collection time.

FIG. 21 shows the Raman spectra of various fragments obtained by trypsindigestion of serum protein. Peptides were separated by reverse-phasehigh pressure liquid chromatography (HPLC) on a C18 column.

DETAILED DESCRIPTION

Definitions

As used herein, “a” or “an” may mean one or more than one of an item.

The terms “protein,” “polypeptide” and “peptide” refer to polymericmolecules assembled in linear fashion from amino acids. The distinctionbetween the terms is primarily one of length, with peptides typicallyranging from about 2 to about 25 amino acid residues, polypeptides fromabout 10 to about 100 amino acid residues and proteins about 50 residuesor longer. The terms overlap and the skilled artisan will realize thatwhere the following disclosure refers to proteins or polypeptides orpeptides, the terms encompass polymers of any length. Where the presentspecification uses the term “protein”, it will be understood that theterm also encompasses “polypeptide” and/or “peptide”. It is contemplatedthat proteins to be analyzed may comprise naturally occurring amino acidresidues, modified amino acid residues, derivatized amino acid residues,amino acid analogues and/or non-naturally occurring amino acids. Aminoacid residues that have been labeled with any labels are alsoencompassed. Although amino acid residues in naturally occurringproteins are typically joined together by peptide bonds, within thescope of the disclosed methods amino acid residues may be joined bypeptide bonds or by any other type of known covalent attachment.

The terms “nanopore”, “nanochannel” and “nanotube” refer respectively toa hole, channel or tube with a diameter or width of between 1 and 999nanometers (nm), more typically between 1 and 100 nm, even moretypically between 1 and 10 nm. As used herein, the terms “nanopore”,“nanotube” and “nanochannel” may be used interchangeably. The skilledartisan will realize that where the specification refers to a“nanopore,” different alternatives may use a “nanochannel” or“nanotube.” The only requirement is that the nanopore, nanochannel ornanotube connect one fluid filled compartment to another and allow thepassage and detection of labeled proteins.

As used herein, “operably coupled” means that there is a functionaland/or structural relationship between two or more units. For example, adetector may be “operably coupled” to a nanopore if the detector isarranged so that it may identify labeled amino acid residues passingthrough the nanopore. Similarly, a nanopore may be operably coupled to achamber if proteins in the chamber can pass through the nanopore. Adetector may also be “operably coupled” to a nanopore where the detectorand/or sensing elements of the detector are integrated into thenanopore.

As used herein, “fluid communication” refers to a functional connectionbetween two or more compartments that allows fluids to pass between thecompartments. For example, a first compartment is in “fluidcommunication” with a second compartment if fluid may pass from thefirst compartment to the second and/or from the second compartment tothe first compartment.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed methods and apparatus are of use for the rapid, automatedsequencing and/or identification of proteins. Advantages over prior artmethods include high throughput, sensitive detection of single labeledprotein molecules, nanometer scale resolution of amino acid residuedistances and lower unit cost of protein sequencing and/oridentification.

The following detailed description contains numerous specific details inorder to provide a more thorough understanding of the claimed methodsand apparatus. However, it will be apparent to those skilled in the artthat the methods and apparatus may be practiced without these specificdetails. In other instances, devices, methods, procedures, andindividual components that are well known in the art have not beendescribed in detail herein.

As illustrated in FIG. 1, a nucleic acid template may be placed in oneor more chambers 120, each chamber 120 to contain a different labeledamino acid. Labeled proteins encoded by the nucleic acid template may beproduced by in vitro translation or by linked transcription/translation.The labeled proteins may pass through one or more nanopores associatedwith each chamber, the nanopores permeating one or more sensor layersoperably coupled to a detector. As a labeled protein passes through ananopore, labeled amino acid residues are detected. The distancesbetween labeled amino acid residues are determined and a distance map140 is compiled for each type of labeled amino acid residue. Thedistance maps 140 may be used to sequence 150 and/or identify 160 thelabeled protein.

The skilled artisan will realize that the distance maps 140 ofconsideration may show distances in the sub-nanometer or greater scale.For example, a single amino acid in a linear protein sequence would havea size of about 0.6 nm. During typical gel electrophoresis of proteins(field strength of about 10 volt/cm), molecules may travel about 100 mmin 60 minutes (or about 28,000 nm per second). Since currently availableelectrical detectors are capable of counting down to the femto secondscale, detection of adjacent amino acids is well within the detectionlimits. Given the mobility rate of proteins under electrophoresis, a 1nanosecond time frame would be equivalent to a distance of 0.036 nm,which is less than the carbon-carbon bond length of about 0.154 nm. Itwould take about 20 nanoseconds to detect two adjacent amino acidresidues. The distance maps 140 may range from the average subunitdistance (0.6 nm) up to the length of a full-length protein, which maybe thousands of amino acids long.

In alternative methods, labeled proteins may be prepared by incubatingcells in, for example, a solution comprising labeled amino acid andpurifying one or more proteins from the incubated cells. In otheralternative, cells may be transformed with an expression vector encodinga protein of interest and allowed to form labeled proteins. Where twentychambers 120 are used containing all twenty different labeled amino acidresidues, the distance maps 140 may be compiled into a complete proteinsequence 150.

Proteins, Polypeptides and Peptides

Proteins to be analyzed may be: [1] purified from natural sources; [2]expressed by in vitro translation of an mRNA species or by linkedtranscription/translation of a DNA species; and/or [3] expressed in ahost cell that has been transformed with a gene or a complementary DNA(cDNA) species. These methods are not limiting and proteins to beanalyzed may be prepared by any method known in the art.

Protein Purification

Proteins to be analyzed may be partially or fully purified from avariety of sources before analysis. Protein purification techniques arewell known in the art. These techniques typically involve an initialcrude fractionation of cell or tissue homogenates and/or extracts intoprotein and non-protein fractions. Fractionation may utilize, forexample, differential solubility in aqueous solutions, detergents and/ororganic solvents, elimination of classes of contaminants such as nucleicacids by enzymatic digestion, precipitation of proteins with ammoniumsulphate, polyethylene glycol, antibodies, heat denaturation and thelike, followed by ultracentrifugation. A variety of detergents of use inprotein purification are known in the art, including but not limited toionic surfactants (e.g., sodium dodecyl sulphate, sodium cholate, sodiumdeoxycholate, hexadecyltrimethylammonium bromide) and non-ionicsurfactants (e.g., Triton X-100, Tween-20, Brij-35, digitonin, Nonidet®P40, octylglucoside). Non-ionic detergents may be of greater use whereelectrical detection of tagged residues is used. For optical detection,either ionic or non-ionic detergents may be of use. A detergent thatdoes not exhibit substantial absorption and/or emission at thewavelengths used for excitation and detection would be of greater usefor optical detection. Reducing agents such as dithiothreitol orβ-mercaptoethanol may be of use to reduce disulfide bonds and dissociateprotein aggregates. Low molecular weight contaminants may be removed bydialysis, filtration and/or organic phase extraction.

Protein(s) of interest may be purified using chromatographic and/orelectrophoretic techniques to achieve partial or complete purification.Methods suited to the purification of proteins, polypeptides andpeptides include, but are not limited to, ion-exchange chromatography,gel exclusion chromatography, polyacrylamide gel electrophoresis,affinity chromatography, immunoaffinity chromatography, hydroxylapatitechromatography, hydrophobic interaction chromatography, reverse phasechromatography, isoelectric focusing, fast protein liquid chromatography(FPLC) and high pressure liquid chromatography (HPLC). These and othermethods of protein purification are known in the art and are notlimiting for the claimed subject matter. Any known method of proteinpurification may be used. There is no requirement that the protein mustbe in its most purified state. Methods exhibiting a lower degree ofrelative purification may, for example, have advantages in increasedrecovery of labeled protein.

Affinity chromatography may be used for purification of some proteins.The method relies on an affinity between a protein and a molecule towhich it can specifically bind. Chromatography material may be preparedby covalently attaching a protein-binding ligand, such as an antibody,antibody fragment, receptor protein, substrate, inhibitor, product or ananalog of such ligands to an insoluble matrix, such as columnchromatography beads or a nylon or other membrane. The matrix may thenbe used to specifically adsorb the target protein from a solution.Elution occurs by changing the solvent conditions (e.g. pH, ionicstrength, temperature, detergent concentration, etc.). One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. Methods for generating antibodies against various typesof proteins for use in immunoaffinity chromatography are well known inthe art.

Proteins of interest may be specifically labeled in order to facilitatepurification. The protein of interest may be followed through apurification protocol by looking for the presence of the label. Proteinsmay be post-translationally labeled using side chain specific and/orselective reagents as discussed below. Various methods for proteinlabeling are known in the art, discussed in more detail below.

In Vitro Translation

Proteins may be expressed using an in vitro translation system with mRNAtemplates. Complete kits for performing in vitro translation areavailable from commercial sources, such as Ambion (Austin, Tex.),Promega (Madison, Wis.), Amersham Pharmacia Biotech (Piscataway, N.J.),Invitrogen (Carlsbad, Calif.) and Novagen (Madison, Wis.). Such kits mayutilize total RNA, purified polyadenylated mRNA, and/or purifiedindividual mRNA species obtained from a cell, tissue or other sample.Methods of preparing different RNA fractions and/or individual mRNAspecies for use in in vitro translation are known. (E.g., Sambrook, etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al.,Current Protocols in Molecular Biology, Wiley and Sons, New York, N.Y.,1994).

Commonly used in vitro translation systems are based on rabbitreticulocyte lysates, wheat germ extracts and E. coli extracts. In vitrotranslation systems based on rabbit reticulocyte lysates areparticularly robust and efficient for eukaryotic translation. Thesystems contain crude cell extracts including ribosomal subunits,transfer RNAs (tRNAs), aminoacyl-tRNA synthetases, initiation,elongation and termination factors and/or all other components requiredfor translation. The natural amino acids present in such extracts may besupplemented with one or more different types of labeled amino acids.Depending on the application, the label may be restricted to a singletype of amino acid. Alternatively, a sample to be translated may bedivided up into different sub-samples, each of which may be exposed to adifferent type of labeled amino acid. For optical detection methods,tryptophan or 5-fluoro-tryptophan exhibit natural fluorescence and maybe used for Raman spectroscopy. Labels may be added to other amino acidresidues either before protein synthesis or by post-translationalmodification. Other components of use in supplementing in vitrotranslation systems and methods of use of such systems are known in theart (see, e.g., Ambion website).

In vitro translation may be linked to transcription of genes to generatemRNAs. Such linked transcription/translation systems may use PCR®amplification products and/or DNA sequences inserted into standardexpression vectors such as BACs (bacterial artificial chromosomes), YACs(yeast artificial chromosomes), cosmids, plasmids, phage and/or otherknown expression vectors. Linked transcription/translation systems areavailable from commercial sources (e.g., Proteinscript™ II kit, Ambion,Austin, Tex.; Quick Coupled System, Promega, Madison, Wis.; Expressway,Invitrogen, Carlsbad, Calif.). Such systems may incorporate variouselements to optimize the efficiency of transcription and translation,such as polyadenylation sequences, consensus ribosomal binding (Kozak)sequences, Shine-Dalgarno sequences and/or other regulatory sequencesknown in the art.

Labeled proteins may be purified from the crude in vitro translationmixture prior to analysis, or alternatively may be analyzed withoutpurification. The use of protein purification may depend in part onwhether a crude RNA fraction or a purified RNA species is used as thetemplate for translation.

Protein Expression in Host Cells

Nucleic acids encoding target proteins of interest may be incorporatedinto expression vectors for transformation into host cells andproduction of the encoded proteins. Non-limiting examples of host celllines known in the art include bacteria such as E. coli, yeast such asPichia pastoris, and mammalian cell lines such as VERO cells, HeLacells, Chinese hamster ovary cell lines, human embryonic kidney (HEK)293 cells, mouse neuroblastoma N2A cells, or the W138, BHK, COS-1,COS-7, 293, HepG2, 3T3, RIN, L-929 and MDCK cell lines. These and otherhost cell lines may be obtained from standard sources, such as theAmerican Type Culture Collection (Rockville, Md.) or commercial vendors.

A complete gene can be expressed or fragments of a gene encodingportions of a protein can be expressed. The gene or gene fragmentencoding protein(s) of interest may be inserted into an expressionvector by standard cloning techniques. Expression libraries containingpart or all of the messenger RNAs expressed in a given cell or tissuetype may be prepared by known techniques. Such libraries may be screenedfor clones encoding particular proteins of interest, for example usingantibody or oligonucleotide probes and known screening techniques.

The engineering of DNA segment(s) for expression in a prokaryotic oreukaryotic system may be performed by techniques generally known in theart. Any known expression system may be employed for protein expression.Expression vectors may comprise various known regulatory elements forprotein expression, such as promoters, enhancers, ribosome bindingsites, termination sequences, polyadenylation sites, etc.

Promoters commonly used in bacterial expression vectors include theβ-lactamase, lactose and tryptophan promoter systems. Suitable promotersequences in yeast expression vectors include the promoters for3-phosphoglycerate kinase or other glycolytic enzymes. Promoters of usefor mammalian cell expression may be derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter or the early and latepromoters of SV40). Many other promoters are known and may be used inthe practice of the disclosed methods.

Eukaryotic expression systems of use include, but are not limited to,insect cell systems infected with, for example, recombinant baculovirus,or plant cell systems infected with recombinant cauliflower mosaic virusor tobacco mosaic virus. In an exemplary insect cell system, Autographacalifornica nuclear polyhidrosis virus is used as a vector to expressforeign genes in Spodoptera frugiperda cells or the Hi5 cell line(Invitrogen, Carlsbad, Calif.). Nucleic acid coding sequences are clonedinto, for example, the polyhedrin gene of the virus under control of thepolyhedrin promoter. Recombinant viruses containing the cloned gene arethen used to infect Spodoptera frugiperda cells and the inserted gene isexpressed (e.g., U.S. Pat. No. 4,215,051; Kitts et al., Biotechniques14:810-817, 1993; Lucklow et al., J. Virol., 67:4566-79, 1993). Otherexemplary insect cell expression vectors are based on baculovirusvectors, for example, pBlueBac (Invitrogen, Sorrento, Calif.).

An exemplary expression system in mammalian cell lines may utilizeadenovirus as an expression vector. Coding sequences may be ligated to,e.g., the adenovirus late promoter. The cloned gene may be inserted intothe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3)results in a recombinant virus that is capable of infecting andexpressing cloned proteins in mammalian host cells. The disclosedexamples are not limiting and any known expression vector may be used.

Cells transformed with expression vectors may be selected fromnon-transformed cells. A number of selection systems may be used,including but not limited to, the thymidine kinase gene,hypoxanthine-guanine phosphoribosyltransferase gene, methotrexateresistance gene, neomycin phosphotransferase gene and hygromycinresistance gene. These genes, contained in standard cloning vectors,either confer resistance to cytotoxic agents or allow cell growth innutrient deficient medium.

Expressed proteins may be partially or completely purified beforeanalysis. Protein purification may be facilitated by expressing clonedsequences as fusion proteins containing short leader sequences thatallow rapid affinity purification. Examples of such fusion proteinexpression systems are the glutathione S-transferase system (Pharmacia,Piscataway, N.J.), the maltose binding protein system (NEB, Beverley,Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×His system(Qiagen, Chatsworth, Calif.). A leader sequence may be linked to aprotein by a specific recognition site for a protease, allowing removalof the leader sequence prior to protein analysis. Examples of suitableprotease recognition sequences include those recognized by the TobaccoEtch Virus protease (Life Technologies, Gaithersburg, Md.) or Factor Xa(New England Biolabs, Beverley, Mass.). Alternatively, expressedproteins may be purified by standard techniques discussed above.

Although the methods disclosed above are directed towards analysis ofproteins, they are also applicable to the analysis of other types ofanalytes. For example, cells could be incubated in a labeledmonosaccharide and polysaccharides could be purified and identifiedand/or sequenced as described herein. The labeled subunit (e.g.,monosaccharide) may be derivatized to prevent its metabolism andconversion to a different structure. Subunits and polymeric forms ofsuch analytes are known in the art.

Protein Labeling

Proteins to be analyzed may comprise labeled amino acid residues. Aminoacids may be labeled by any methods known in the art. A labeled aminoacid residue may be incorporated into a protein during synthesis.Alternatively, labels may be attached to amino acid residues by covalentor non-covalent bonding after protein synthesis.

Labels of use in the disclosed methods may include, but are not limitedto, any composition detectable by electrical, optical,spectrophotometric, photochemical, biochemical, immunochemical, and/orchemical techniques. Labels may include, but are not limited to,conducting, luminescent, fluorescent, chemiluminescent, bioluminescentand phosphorescent labels, nanoparticles, metal nanoparticles, goldnanoparticles, silver nanoparticles, chromogens, antibodies, antibodyfragments, genetically engineered antibodies, enzymes, substrates,cofactors, inhibitors, binding proteins, magnetic particles and spinlabels.

Non-limiting examples of photodetectable labels that may be used includedansyl chloride, rhodamine isothiocyanate, TRIT (tetramethyl rhodamineisothiol), NBD (7-nitrobenz-2-oxa-1,3-diazole), Texas Red, phthalicacid, terephthalic acid, isophthalic acid, cresyl fast violet, cresylblue violet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine,biotin, digoxigenin, fluorescein,5-carboxy-4′,5′-dichloro-2′,7′-dimethoxy fluorescein,5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein,5-carboxyrhodamine, aminoacridine, 6-carboxyrhodamine,6-carboxytetramethyl amino phthalocyanines, azomethines, cyanines,xanthines, succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allophycocyanin, phycocyanin C,phycocyanin R, thiamine, phycoerythrocyanin, phycoerythrin R, luciferin,or acridinium esters. These and other luminescent labels may be obtainedfrom commercial sources such as Molecular Probes (Eugene, Oreg.) andattached to amino acids by methods known in the art. Alternatively,certain pre-labeled amino acids are commercially available (e.g.,Molecular Probes, Eugene, Oreg.).

Amino acid residues may be labeled with electrically detectable labels,such as metal nanoparticles. Gold or silver nanoparticles of between 1nm and 3 nm in size may be used, although nanoparticles of differentdimensions and mass may also be used. Methods of preparing nanoparticlesare known. (See e.g., U.S. Pat. Nos. 6,054,495; 6,127,120; 6,149,868;Lee and Meisel, J. Phys. Chem. 86:3391-3395, 1982.) Nanoparticles mayalso be obtained from commercial sources (e.g., Nanoprobes Inc.,Yaphank, N.Y.; Polysciences, Inc., Warrington, Pa.). Modifiednanoparticles are available commercially, such as Nanogold®nanoparticles from Nanoprobes, Inc. (Yaphank, N.Y.). Nanogold®nanoparticles may be obtained with either single or multiple maleimide,amine or other groups attached per nanoparticle. The Nanogold®nanoparticles also are available in either positively or negativelycharged form. Such modified nanoparticles may be attached covalently toamino acid residues either before or after the amino acid residues areincorporated into proteins. Nanoparticles or other labels may beattached to amino acid residues via any known linker compound to reducesteric hindrance and facilitate protein polymerization.

Labeled amino acid residues may be incorporated into proteins made froma nucleic acid template. Alternatively, labels may be attached to aparticular type of amino acid residue after synthesis of the protein. Insome methods, the label may be attached by antibody-antigeninteractions. A label such as fluorescein or biotin may be attached toone end of a protein molecule, such as the N-terminal or C-terminal end.

Proteins may be post-translationally labeled using side-chain specificand/or selective reagents. Such reagents and methods forpost-translational modification are known in the art. Non-limitingexemplary reagents that may be used include acetic anhydride (lysine,cysteine, serine and tyrosine); trinitrobenzenesulfonate (lysine);carbodiimides (glutamate, aspartate); phenylglyoxal (arginine);2,3-butanedione (arginine); pyridoxal phosphate (lysine);p-chloromercuribenzoate (cysteine); 5,5′-dithiobis(2-nitro-benzoic acid)(cysteine); diethylpyrocarbonate (lysine, histidine); N-bromosuccinimide(tryptophan) and tetranitromethane (cysteine, tyrosine). Such reagentsmay be modified to attach various types of labels, such as Raman labels.Alternatively, Raman labels and/or gold nanoparticles that containreactive groups for attachment to various types of amino acid sidechains may be obtained from commercial sources (Molecular Probes,Eugene, Oreg.; Nanoprobes, Inc., Yaphank, N.Y.).

Various cross-linking reagents known in the art, such ashomo-bifunctional, hetero-bifunctional and/or photoactivatablecross-linking reagents may be used to attach labels to proteins.Non-limiting examples of such reagents include bisimidates;1,5-difluoro-2,4-(dinitrobenzene); N-hydroxysuccinimide ester of subericacid; disuccinimidyl tartarate; dimethyl-3,3′-dithio-bispropionimidate;N-succinimidyl-3-(2-pyridyldithio)propionate;4-(bromoaminoethyl)-2-nitrophenylazide; and 4-azidoglyoxal. Methods ofuse of cross-linking reagents are well known in the art.

Nanopores, Nanochannels and Nanotubes

Labeled proteins or other polymers may be passed through one or morenanopores, nanochannels or nanotubes for analysis. As used herein, theterms nanopores, nanotubes and nanochannels are used interchangeably.The skilled artisan will realize that where the specification refers toa nanopore, different alternatives may use a nanochannel or nanotube.The only requirement is that the nanopore, nanochannel or nanotubeconnect one fluid filled compartment to another and allow the passageand detection of labeled proteins.

Size Characteristics

Nanopores of use may be about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nm indiameter. However, the diameter may range between 1-3, 1-5, 1-10, 1-20,1-50, 1-100, 5-10, 5-20, 10-20, 20-50, 30-75, 50-75, 50-100, 75-100,100-300, 300-400, 400-500 or 100-999 nm. Nanopore diameter may beselected to pass a single protein at a time, in a linear configuration.In certain alternatives, nanopore diameter may be selected to be toosmall to pass a protein in a globular or folded conformation. Thedimensions of various folded and unfolded proteins are known in the artand may be estimated by known techniques, such as filtration orultracentrifugation. Proteins to be analyzed may be unfolded and/orpartially or fully denatured by known methods to facilitate theirpassage through a nanopore in a linear conformation. Such methods mayinclude, but are not limited to, exposure to media of either alkaline oracidic pH, use of high or low salt concentrations, use of detergentssuch as sodium dodecyl sulphate, octylglucoside or Triton X-100, use ofchaotrophic agents such as urea or guanidinium, treatment with disulfidereducing agents such as dithiothreitol or mercaptoethanol, exposure toorganic solvents, etc. Alternatively, linear peptides may be generatedby limited proteolytic digestion of proteins. Where the amino acidresidues are to be labeled with bulky groups, the nanopores may belarger to allow passage of labeled proteins. In alternatives thatutilize nanotubes or nanochannels in place of nanopores, the same sizeconsiderations apply to the diameter or width of the nanotubes ornanochannels.

Fabrication of Nanopores, Nanotubes and Nanochannels

Fabrication of nanopores, nanotubes and/or nanochannels, individually orin arrays, may utilize any technique known in the art for nanoscalemanufacturing. Nanopores, nanochannels and/or nanotubes may beconstructed on a solid-state matrix comprising sensor layers using knownnanolithography methods, including but not limited to chemical vapordeposition, electrochemical deposition, chemical deposition,electroplating, thermal diffusion and evaporation, physical vapordeposition, sol-gel deposition, focused electron beam, focused ion beam,molecular beam epitaxy, dip-pen nanolithography, reactive-ion beametching, chemically assisted ion beam etching, microwave assisted plasmaetching, electro-oxidation, scanning probe methods, chemical etching,laser ablation, or any other method known in the art (E.g., U.S. Pat.No. 6,146,227).

Nanopores, nanotubes and/or nanochannels may penetrate one or moresensor layers. The sensor layers may comprise semiconductor materialsincluding, but not limited to, silicon, silicon dioxide, siliconnitride, germanium, gallinium arsenide, and/or metal-based compositionssuch as metals or metal oxides. Sensor layers may be processed byelectronic beam, ion beam and/or laser lithography and etching to createa channel, groove, or hole. Conducting layers comprising metals may bedeposited onto a semiconductor surface by means of field evaporationfrom a scanning tunnel microscope (STM) or atomic force microscope (AFM)tip or from a solution. Insulating layers may be formed by oxidizing thesemiconductor's surface to an insulating composition.

Channels or grooves may be etched into a semiconductor surface byvarious techniques known in the art including, but not limited to,methodologies using an STM/AFM tip in an oxide etching solution. Afterchannels are formed, two semiconductor surfaces may be opposed to createone or more nanopores or nanochannels that penetrate the semiconductor.STM tip methodologies may be used to create nanopores, nanodetectors,nanowires, nanoleads, nanochannels, and other nanostructures usingtechniques known in the art. Scanning probes, chemical etchingtechniques, and/or micromachining may be used to cutmicrometer-dimensioned or nanometer-dimensioned channels, grooves orholes in a semiconductor substrate.

Nano-molding may be employed, wherein formed nanotubes, such as carbonor metallic nanotubes, are placed or grown on a semiconductor chipsubstrate. After depositing additional material on the substrate, thenanotubes are removed, leaving a nanochannel and/or nanopore imprint inthe substrate material. Such nanostructures can be built in clusterswith properties of molecular electrodes that may function as detectors.

Nanopores and/or nanochannels may be made using a high-throughputelectron-beam lithography system. Electron-beam lithography may be usedto write features as small as 5 nm on silicon chips. Sensitive resists,such as polymethyl-methacrylate, coated on silicon surfaces may bepatterned without use of a mask. The electron-beam array may combine afield emitter cluster with a microchannel amplifier to increase thestability of the electron beam, allowing operation at low currents. TheSoftMask™ control system may be used to control electron-beamlithography of nanoscale features on a semiconductor chip substrate.

Nanopores and/or nanochannels may be produced using focused atom lasers(e.g., Bloch et al., “Optics with an atom laser beam,” Phys. Rev. Lett.87:123-321,1). Focused atom lasers may be used for lithography, muchlike standard lasers or focused electron beams. Such techniques arecapable of producing micron scale or even nanoscale structures on achip. In other alternatives, dip-pen nanolithography may be used to formnanochannels (e.g., Ivanisevic et al., “Dip-Pen Nanolithography onSemiconductor Surfaces,” J. Am. Chem. Soc., 123: 7887-7889,1). Dip-pennanolithograpy uses AFM techniques to deposit molecules on surfaces,such as silicon chips. Features as small as 15 nm in size may be formed,with spatial resolution of 10 nm. Nanoscale pores and/or channels may beformed by using dip-pen nanolithography in combination with regularphotolithography techniques. For example, a micron scale line in a layerof resist may be formed by standard photolithography. Using dip-pennanolithography, the width of the line and the corresponding diameter ofthe channel after etching may be narrowed by depositing additionalresist compound. After etching of the thinner line, a nanoscale channelmay be formed. Alternatively, AFM methods may be used to removephotoresist material to form nanometer scale features.

Ion-beam lithography may be used to create nanopores and/or nanochannelson a chip (e.g., Siegel, “Ion Beam Lithography,” VLSI Electronics,Microstructure Science, Vol. 16, Einspruch and Watts Eds., AcademicPress, New York, 1987). A finely focused ion beam may be used to writenanoscale features directly on a layer of resist without use of a mask.Alternatively, broad ion beams may be used in combination with masks toform features as small as 100 nm in scale. Chemical etching, forexample, with hydrofluoric acid, may be used to remove exposed siliconor other chip material that is not protected by resist. The skilledartisan will realize that the techniques disclosed above are notlimiting, and that nanopores and/or nanochannels may be formed by anymethod known in the art.

The surfaces of nanopores, nanotubes or nanochannels may be modified bycoating, for example to transform a surface from a hydrophobic to ahydrophilic surface and/or to decrease adsorption of polymers such asproteins to a surface. Surface modification of common chip materialssuch as glass, silicon and/or quartz is known in the art (e.g., U.S.Pat. No. 6,263,286). Such modifications may include, but are not limitedto, coating with commercially available capillary coatings (Supelco,Bellafonte, Pa.), silanes with various functional groups such aspolyethyleneoxide or acrylamide, or any other known coating. Suchcoatings may not be appropriate where they would interfere with labeldetection, such as interfering with electrical conductivity using anelectrical detector.

Carbon Nanotubes

Nanopores may comprise, be attached to or be replaced by nanotubes, suchas carbon nanotubes. Carbon nanotubes may be coated with an organic orinorganic composition, leaving a deposited layer “mold” on the carbonnanotube. When the nanotube is removed and separated from the organic orinorganic deposit, a nanopore or nanochannel may be created in the“mold.” Carbon nanotubes may be formed in a semiconductor with othercomponents, such as sensor layers, formed around the nanotubes.

Carbon nanotubes may be manufactured by chemical vapor deposition (CVD),using ethylene and iron catalysts deposited on silicon (e.g., Cheung etal. PNAS 97:3809-3813, 2000). Single-wall carbon nanotubes may be formedon silicon chips by CVD using AFM Si₃N₄ tips (e.g., Cheung, et al.,2000; Wong, et al. Nature 394: 52-55, 1998). A flat surface of 1-5 μm²may be created on the silicon AFM tips by contact with silicon or CVDdiamond surfaces (GE Suprabrasives, Worthington, Ohio) at high load (˜1μN), at high scan speed (30 Hz), and with a large scan size (40 μm) forseveral minutes. Approximately 100 nm diameter, 1 μm deep pores in theends of the AFM tips may be made by anodization at 2.1 V for 100 sec.Anodized tips may be etched in 0.03% KOH in water for 50 sec, afterwhich excess silicon may be removed with ethanol, resulting in nanoporesformed at the surface of the tip.

Carbon nanotubes may be attached to AFM tips using known methods. Forexample, iron catalyst consisting of iron oxide nanoparticles may besynthesized according to Murphy et al. (Austr. J. Soil Res. 13:189-201,1975). Iron catalyst (0.5 to 4 nm particles) may be electrochemicallydeposited from a colloidal suspension into the pores using platinumcounter electrodes at −0.5 V (Cheung, et al., 2000). Tips may be washedin water to remove excess iron oxide particles. AFM tips may be oxidizedby heating in oxygen gas and carbon nanotubes may be grown on thecatalyst by controlled heating and cooling in the presence of a carbonsource (Murphy et al., 1975; Cheung et al., 2000). The diameter of theresulting nanotubes should correspond to the size of the iron oxidecatalyst used (0.5 to 4 nm). Individual, single-walled nanotubesprepared under these conditions are aligned perpendicular to theflattened surface of the AFM tip. Residual iron catalyst may be removedby known methods.

Nanotubes may be cut to a predetermined length using known techniques.In some embodiments of the invention, carbon nanotubes may be attachedto pyramids of gold-coated silicon cantilevers using an acrylicadhesive. The carbon nanotubes may be shortened to a defined length byapplication of a bias voltage between the tip and a niobium surface inan oxygen atmosphere (Wong, et al., Nature 394:52-55, 1998).Alternatively, high-energy beams may be used to shorten carbonnanotubes. Such high energy beams may include, but are not limited to,laser beams, ion beams, and electron beams. Alternative methods fortruncating carbon nanotubes are known in the art (e.g., U.S. Pat. No.6,283,812). Preformed carbon nanotubes may be attached to a chipmaterial such as silicon, glass, ceramic, germanium, polystyrene, and/orgallium arsenide (e.g., U.S. Pat. Nos. 6,038,060 and 6,062,931).

A first set of carbon nanotubes may be used as cold cathode emitters onsemiconductor chips, associated with a second set of nanotubescontaining proteins. The first set of nanotubes may be used to createlocal electrical fields of at least 10⁶ volts/cm, when an externalvoltage of between 10 and 50 volts is applied. Such an electric field inthe first set of nanotubes can be used to drive proteins through thesecond set of nanotubes, or to generate an electrical or electromagneticsignal to detect labeled amino acid residues (Chuang, et al., 2000; U.S.Pat. No. 6,062,931). Electromagnetic radiation from a third set ofnanotubes may excite a luminescent label attached to a protein passingthrough a second set of nanotubes, leading to emission of light detectedby a photodetector that is operably coupled to a first set of nanotubes.

Ion Channels on Semiconductor Chips

Nanopores may comprise single ion channels in lipid bilayer membranes(e.g., Kasianowitz, et al., Proc. Natl. Acad. Sci. USA 93:13770-13773,1996). Such ion channels may include, but are not limited to,Staphylococcus aureus alpha-hemolysin and/or mitochondrialvoltage-dependent anion channels. These ion channels may remain open forextended periods of time. An electric field applied to proteins cancause these molecules to move through ion channels in lipid bilayermembranes. Ion channels may be incorporated into chips and operablycoupled to detectors.

Micro-Electro-Mechanical Systems (MEMS)

Nanopores, sensor layers and other components of the disclosed apparatusmay be incorporated into one or more Micro-Electro-Mechanical Systems(MEMS). MEMS are integrated systems that may comprise mechanicalelements, actuator elements, control elements, detector elements and/orelectronic elements. All of the components may be manufactured by knownmicrofabrication techniques on a common chip, comprising a silicon-basedor equivalent substrate (e.g., Voldman et al., Ann. Rev. Biomed. Eng.1:401-425, 1999).

The electronic components of MEMS may be fabricated using integratedcircuit (IC) processes (e.g., CMOS, Bipolar, or BICMOS processes). Theymay be patterned using photolithographic and etching methods known forsemiconductor chip manufacture. The micromechanical components may befabricated using “micromachining” processes that selectively etch awayparts of the silicon wafer and/or add new structural layers to form themechanical and/or electromechanical components. Basic techniques in MEMSmanufacture include depositing thin films of material on a substrate,applying a patterned mask on top of the films by photolithographicimaging or other known lithographic methods, and selectively etching thefilms. A thin film may have a thickness in the range of a few nanometersto 100 micrometers. Deposition techniques of use may include chemicalprocedures such as chemical vapor deposition (CVD), electrodeposition,epitaxy and thermal oxidation and physical procedures like physicalvapor deposition (PVD) and casting. Sensor layers, of 5 nm thickness orless may be formed by such known techniques. Standard lithographytechniques may be used to create sensor layers of micron or sub-microndimensions, operably coupled to detectors and nanopores.

The manufacturing method is not limiting and any methods known in theart may be used, such as atomic layer deposition, pulsed DC magnetronsputtering, vacuum evaporation, laser ablation, injection molding,molecular beam epitaxy, dip-pen nanolithograpy, reactive-ion beametching, chemically assisted ion beam etching, microwave assisted plasmaetching, focused ion beam milling, electron beam or focused ion beamtechnology or imprinting techniques. Methods for manufacture ofnanoelectromechanical systems may be used. (See, e.g., Craighead,Science 290:1532-36,0.)

It is contemplated that some or all of the components of the apparatusmay be constructed as part of an integrated MEMS device. Nanoelectrodescomprising conducting metals such as gold, platinum, or copper may beoperably coupled to nanopores, nanochannels and/or nanotubes using STMtechnologies known in the art (e.g., Kolb et al., Science 275:1097-1099,1997). Nanoelectrodes, detectors and other components may be connectedby nanowires.

Detectors

Photodetectors

Amino acid residues labeled with photolabels may be detected using anexcitatory light source and a photodetector (e.g., Sepaniak et al., J.Microcol. Separations 1:155-157, 1981; Foret et al., Electrophoresis7:430-432, 1986; Horokawa et al., J. Chromatog. 463:39-49 1989; U.S.Pat. No. 5,302,272). Exemplary light sources include diode-lasers,vertical cavity surface-emitting lasers, edge-emitting lasers, surfaceemitting lasers and quantum cavity lasers, for example a ContinuumCorporation Nd-YAG pumped Ti:Sapphire tunable solid-state laser and aLambda Physik excimer pumped dye laser. Exemplary photodetectors includephotodiodes, avalanche photodiodes, photomultiplier tubes, multianodephotomultiplier tubes, phototransistors, vacuum photodiodes, siliconphotodiodes, fiber-optic or phototransistor detectors and charge-coupleddevices (CCDs). An avalanche photodiode (APD) may be used to detect lowlight levels. The APD process uses photodiode arrays for electronmultiplication effects (U.S. Pat. No. 6,197,503).

A photodetector, light source and nanopore may be fabricated into asemiconductor chip using known N-well Complementary Metal OxideSemiconductor (CMOS) processes (Orbit Semiconductor, Sunnyvale, Calif.).Alternatively, the detector, light source and nanopore may be fabricatedin a silicon-on-insulator CMOS process (e.g., U.S. Pat. No. 6,117,643).In other alternatives, an array of diode-laser illuminators and CCDdetectors may be placed on a semiconductor chip (U.S. Pat. Nos.4,874,492 and 5,061,067; Eggers et al., BioTechniques 17: 516-524,1994).

A photodetector may be positioned perpendicular to a light source tominimize background light. The light source may be optically separatedfrom the photodetector by one or more light opaque layers. Photonsgenerated by excitation of luminescent labels may be collected by afiber optic and transferred to a CCD detector on a chip (e.g., U.S. Pat.No. 6,274,320). The times at which labeled amino acid residues aredetected may be recorded and amino acid residue distance maps may beconstructed.

Light sources, such as light-emitting diodes and/or semiconductor lasersmay be incorporated into semiconductor chips (U.S. Pat. No. 6,197,503).Diffractive optical elements that shape a laser or diode light beam mayalso be integrated into a chip. An air-cooled argon laser at 488 nm maybe used to excite fluorescein-labeled proteins. Emitted light may becollected by an optics system comprising a fiber optic, a lens, animaging spectrometer, and a 0° C. thermoelectrically-cooled CCD camera.Alternative examples of photodetectors are known in the art (e.g., U.S.Pat. No. 5,143,8545).

Raman Spectroscopy

Labeled amino acid residues may be detected by Raman spectroscopy. Ramanlabels of use in spectrophotometric detection are well known in the art(e.g., U.S. Pat. Nos. 5,306,403; 6,002,471; 6,174,677). Labeled aminoacid residues may be excited with a laser, photodiode, or other lightsource and the excited amino acid residue detected by a variety of Ramantechniques, including but not limited to surface enhanced Ramanspectroscopy (SERS), surface enhanced resonance Raman spectroscopy(SERRS) normal Raman scattering, resonance Raman scattering, coherentanti-Stokes Raman spectroscopy (CARS), stimulated Raman scattering,inverse Raman spectroscopy, stimulated gain Raman spectroscopy,hyper-Raman scattering, molecular optical laser examiner (MOLE) or Ramanmicroprobe or Raman microscopy or confocal Raman microspectrometry,three-dimensional or scanning Raman, Raman saturation spectroscopy, timeresolved resonance Raman, Raman decoupling spectroscopy or UV-Ramanmicroscopy. In SERS and SERRS, the sensitivity of the Raman detection isenhanced by a factor of 10⁶ or more for molecules adsorbed on roughenedmetal surfaces, such as silver, gold, platinum, copper or aluminumsurfaces. Portions of the nanopores and/or sensor layers may be coatedwith a Raman sensitive metal, such as silver or gold to provide anenhanced Raman signal.

FRET Detection

A protein may also be analyzed using fluorescence resonance energytransfer (FRET). FRET is a spectroscopic phenomenon used to detectproximity between fluorescent donor and acceptor molecules. The donorand acceptor pairs are chosen such that fluorescent emission from thedonor overlaps the excitation spectrum of the acceptor. When the twomolecules are associated at a distance of less than 100 Angstroms, theexcited-state energy of the donor is transferred non-radiatively to theacceptor. If the acceptor molecule is a fluorophore then its emission isenhanced. Compositions and methods for use of FRET are known (e.g., U.S.Pat. No. 5,866,336).

The donor fluorophore molecules may be attached to an amino acidresidue, and the acceptor fluorophore molecules may be connected to ananopore or sensor layer. Following excitation by a light source, thedonor fluorophore molecules may transfer their energy to the acceptormolecules, resulting in an enhanced fluorescent signal from the acceptormolecules that may be detected by a photodetector.

Electrical Detectors

An electrical detector may detect electrical signals from a conductinglayer as a labeled protein passes through a nanopore. Non-limitingexamples of electrical signals include current, voltage, impedance,capacitance, electromotive force, signal sign, frequency or noisesignature measured across a nanopore. An electrical detector may beoperably coupled to one or more conducting layers, a power supply andone or more nanopores penetrating the conducting layers. The detectormay comprise an ammeter, voltmeter, capacitance meter and/orconductivity meter, etc. Other electrical components such as resistorsand/or capacitors may be included in the electrical circuit associatedwith the detector.

In certain methods, first and second buffer chambers may be filled witha low conductivity aqueous buffer. An electrical potential may beapplied to conducting layers flanking a nanopore. When buffer alone ispresent, the resistance between the conducting layers is high. Thepresence of unlabeled regions of proteins passing through the nanoporemay produce a slight increase in conductivity across the nanopore. Thepassage of amino acid residues labeled with highly conductive labels,such as metal nanoparticles, would result in an increase in conductivitythat produces a detectable signal at the detector. The time intervalbetween detectable electrical signals may be measured and used to createa distance map representing the positions of labeled amino acid residueson the protein molecule for each type of labeled amino acid. Thedistance map(s) may be used to identify the protein by comparison withknown protein sequences. By compiling such maps for each of the twentytypes of amino acid residues it would be possible to determine acomplete sequence of the protein.

EXAMPLES Example 1 Apparatus for Protein Identification and/orSequencing

FIG. 2 and FIG. 3 provide non-limiting examples of methods and apparatusfor protein 230, 310 analysis. An apparatus may comprise one or moresub-devices 200, 300. Each sub-device 200, 300 may comprise fluid filledfirst 280, 350 and second 290, 360 chambers, separated by sensor layers212, 323. One or more nanopores 255, 330 may extend through the sensorlayers 212, 323 and allow passage of labeled proteins 230, 310. Thenanopores 255, 330 may be operably coupled to one or more detectors 257,345 that can detect labeled amino acid residues 235, 245, 315 as theypass through the nanopores 255, 330. Electrodes 262, 264, 350, 355 inthe first and second chambers 280, 350, 290, 360 may be used to generatean electrical field that drives labeled proteins 230, 310 from the first280, 350 to the second chamber 290, 360 through the nanopores 255, 330.The electrical gradient may be controlled by a voltage regulator 260,335, which may be operably coupled to a computer 265, 340. The nature ofthe electrical gradient is not limiting and the applied voltage may bealternating current, direct current, pulse field direct current, reversephase current or any other known type of electrical gradient.

Sensor Layer Construction

As illustrated in FIG. 3, photolithography may be used to create anarray of multiplaner structures (0.5×0.5 μm) on a silicon substrate,each structure with a silicon base support and one or more layers ofgold film or other conductive layers 327 separated by one or moreinsulator layers 325 comprising, for example, silicon oxide. Otherinsulator layers 325 overlay the top and bottom conducting layers 327and insulate the sensor layers 323 from the medium in the first 350 andsecond 360 chambers. Conducting and insulating layers 325, 327 may beformed on a chip by standard semiconductor technologies.

A chip containing the multiplanar structures may be divided into two ormore parts. A layer of resist may be coated on the sides of each chippart, perpendicular to the conducting and insulating layers. An AFM/STPtip may be used to etch 5-10 nm lines in the resist layer overlayingeach structure. Chemical etching may be used to create nano-scalegrooves in each of the structures. When the chip parts are aligned andfused together, the grooves form nanopores 330 that extend through thesensor layers 323. Nanowires connecting the conducting layers 327 toelectrical detectors 345 may be formed by known methods discussed above.The nanowires may be used to apply a voltage across the conductinglayers 327. Changes in current, resistance and/or other electricalproperties may be detected with the passage of a protein 310 labeledwith electrically conductive labels, like gold nanoparticles 315,through the nanopore 330. A thin layer of insulating material may beformed on the sides of the divided chip prior to lithography and etchingto prevent current flow except through the nanopore 330.

In alternative devices 200, exemplified in FIG. 2, the sensor layers 212may comprise one or more light opaque layers 215 and photon sensinglayers 220, overlaying a support layer 225, for photodetection of aminoacid residues tagged with photolabels 235, 245. The light opaque layers215 may be formed of any known light opaque material, for example a thinlayer of chrome, silver or gold metal. Similarly, photon sensing layers220 may be comprised of any material that is relatively translucent atthe wavelengths of light emitted by the photolabel 235, 245 for exampleglass, silicon or certain types of plastics.

A wide variety of materials and structures are of use for photon sensinglayers 220. In certain non-limiting examples, the photon sensing layer220 may serve to simply conduct light to the photon sensing elements ofa photodetector 257. In other alternatives, the photon sensing elementmay be integrated into the nanopore 255. For example, a photon sensitivePN junction may be directly fabricated into the photon sensing layer 220surrounding a nanopore 255 by layering with different types of materials(e.g., P-doped and N-doped silicon or gallium arsenide (GaAs)) or bycoating the inner surface of the nanopore 255 with a different type ofsemiconductor material. Methods for forming layers of P-doped andN-doped semiconductors are well known in the arts of computer chipand/or optical transducer manufacture. A photon transducer transduces aphotonic signal into an electrical signal counterpart. Different typesof known photon transducing structures that may be used to detect lightemission include those based on photoconductive materials, photovoltaiccells (photocells), photoemissive materials (photomultiplier tubes,phototubes) and semiconductor pn junctions (photodiodes).

In a photoconductive cell, a semiconductor such as CdS, PbS, PbSe, InSb,InAs, HgCdTe or PbSnTe, behaves like a resistor. The semiconductor is inseries with a constant voltage source and a load resistor. The voltageacross the load resistor is used to measure the resistance of thesemiconductor material. Incident radiation, for example in the form ofan emitted photon from a tagged amino acid residue, causes band-gapexcitation and lowers the resistance of the semiconductor.

A photodiode contains a reverse-bias semiconductor pn junction. Thep-type semiconductor (e.g., boron doped silicon, beryllium doped GaAs)has excess electron holes, while the n-type semiconductor (e.g.,phosphorus doped silicon, silicon doped GaAs) has excess electrons.Under a reverse bias, a depletion layer forms at the pn junction betweenthe p-type and n-type semiconductors. A reverse bias is initiated whenan external electrical potential is applied that forces electron holesin the p-type semiconductor and excess electrons in the n-typesemiconductor to migrate away from the pn junction. When the material isirradiated, electron-hole pairs are formed that move under bias,resulting in a temporary electrical current across the pn junction.Photodiodes and other types of photon transducing structures may beincorporated into a nanopore 255 and used as photon sensing elements ofa photodetector 257.

Polymeric materials may be coated on the chip to enhance signaldetection. Such polymeric materials may include, but are not limited to,polymethylmethacrylate, ultraviolet-curable polyurethanes and epoxies,and other polymers that exhibit optical transparency, low fluorescenceat excitation wavelengths, electrical conductivity and/or insulation.Such materials may be formed into appropriate structures, for example bypolymer casting and chemical or photochemical curing (Kim et al., Nature376: 581-584 1995).

Example 2 Photodetection

As illustrated in FIG. 2, amino acid residues labeled with a photolabel235 may be excited by a light source 210, such as a laser. Excitatorylight may pass through a transparent window 240 in the first chamber280, exciting the photolabel 235 to a higher energy state. The labeledamino acid passes through the light opaque layer 215, cutting off thesource of excitatory light and shielding the photodetector 257 from thelight source 210. As the photolabel 235 passes the photon sensing layer220, it emits a photon and returns to an unexcited state 245. Theemitted photon may be detected by a photodetector 257. The detectedsignal may be amplified by an amplifier 270 and stored and/or processedby a computer 265. The computer 265 may also record the time at whicheach labeled amino acid passes through the nanopore 255, allowing thecalculation of distances between adjacent labeled amino acid residuesand the compilation of a distance map for each type of labeled aminoacid.

In exemplary methods, a highly sensitive cooled CCD detector 257 may beused. The cooled CCD detector 257 has a probability of single-photondetection of up to 80%, a high spatial resolution pixel size (5microns), and sensitivity in the visible through near infrared spectra.(Sheppard, Confocal Microscopy: Basic Principles and System Performancein: Multidimensional Microscopy, Cheng et al. Eds., Springer-Verlag, NewYork, N.Y. pp. 1-51, 1994.) In other examples, a coiledimage-intensified coupling device (ICCD) may be used as a photodetector257 that approaches single-photon counting levels (U.S. Pat. No.6,147,198). A nanochannel plate operates as photomultiplier tube whereina small number of photons triggers an avalanche of electrons thatimpinge on a phosphor screen, producing an illuminated image. Thisphosphor image is sensed by a CCD chip region 257 attached to anamplifier 270 through a fiber optic coupler. A CCD detector 257 on thechip may be sensitive to ultraviolet, visible, and/or infrared spectralight (U.S. Pat. No. 5,846,708).

Example 3 Electrical Detection

As illustrated in FIG. 3, amino acid residues may be tagged with a label315 that can be detected by its electrical properties. In onenon-limiting example, the label 315 may comprise gold nanoparticles 315.As an amino acid residue attached to a gold nanoparticle 315 passesthrough a nanopore 330, it produces detectable changes in theconductivity, resistance and other electrical properties of the nanopore330. The conducting layers 327 flanking a nanopore 330 may be operablycoupled to an electrical detector 345, which may detect any type ofelectrical signal, such as voltage, conductance, resistance,capacitance, etc. The detector 345 may be operably coupled to a computer340 to process and store data. Distance maps showing distances betweenlabeled amino acid residues may be constructed and used to identifyand/or sequence the labeled protein.

A nanopore 2 to 5 nm in diameter may provide fluid communication betweenthe first 350 and second 360 chambers. Proteins 310 labeled with 1 nmgold nanoparticles 315 may be synthesized and/or placed in the firstchamber 350. An electrical detector 345, such as a voltage detector 345,and power supply may be operably coupled to conducting layers 327flanking the nanopore 330. Current across the nanopore 330 may beconverted to voltage and amplified using an AxopatchA (Axon Instruments,Foster City, Calif.) or a Dagan 3900A patch clamp amplifier (DaganInstruments, Minneapolis, Minn.). The signal may be filtered using aFrequency Devices (Haverhill, Mass.) low pass Bessel filter. Data may bedigitized using a National Instruments (Austin, Tex.) AT-MIO-16-X 16-bitboard and LAB WINDOWS/CVI programs. The chip may be shielded fromelectric and magnetic noise sources using a mu-metal box (Amuneal,Philadelphia, Pa.) (see Kasianowicz, et al., 1996).

Example 4 Labeling of Proteins

Exemplary methods for protein labeling are disclosed in FIG. 4 throughFIG. 7. As shown in FIG. 4, cysteine residues may be specifically taggedusing sulfhydryl specific reagents. Naturally occurring cysteineresidues in proteins 410 may be reduced from disulfides through exposureto known thiol reducing agents, such as dithiothreitol orβ-mercaptoethanol. After removal of excess reducing agent, for exampleby ultrafiltration or column chromatography on a gel permeation column,the protein 410 containing reduced cysteine residues may be tagged byusing a thiol specific reagent 420. In one non-limiting example, anacrydite label 420 may be reacted with endogenous cysteines to generatea labeled protein 430. Alternatively, an amine label 430 may beactivated by exposure to N-succinimidyl-4-maleimidobutyrate 450. Theactivating group 450 covalently binds to the amine label 440. Theactivated complex 460 then binds to sulfhydryl groups, resulting information of a labeled protein 470.

FIG. 5. illustrates exemplary methods of labeling proteins 510 on amineresidues, such as lysine, arginine and the N-terminal residue of theprotein 510. As shown in FIG. 5, amine residues on a protein 510 may beactivated by reaction with a reagent 520, such asN-succinimidyl-4-maleimidobutyrate. The activated protein 530 may thenbe reacted with a thiolated label 540, resulting in covalent bondformation and production of a labeled protein 550. In anothernon-limiting example, a water soluble carbodiimide 570, such as EDAC(1-ethyl-3-(3-dimethlaminopropyl)carbodiimide) may be used to cross-linkamine residues on a protein 510 with a carboxylated label 560. Thecarbodiimide 570 binds to and activates the label to form an activatedintermediate 580, which can covalently bond with amine residues. Thecarbodiimide activating group 570 is eliminated and a labeled protein590 is formed.

FIG. 6 shows exemplary methods of protein 610 labeling on carboxylresidues, such as glutamate, aspartate and the C-terminal residue. Aprotein 610 containing carboxyl residues may be activated by reactionwith a water-soluble carbodiimide 615, such as EDAC, and then reactedwith an amine label 625 to form a labeled protein 630. Alternatively,carboxyl residues on a protein 610 may be activated by reaction withboth EDAC 615 and cystamine 635. This results in formation of adisulfide modified protein 640, which can react with an acrydite label645 to form a labeled protein 650. In another alternative, carboxylresidues on a protein 610 may be activated by reaction with EDAC 615 andcystamine 635 and the activated protein 640 reacted with a maleimidelabel 655, resulting in formation of a labeled protein 660.

FIG. 7 illustrates exemplary methods of labeling proteins 710, 745 onserine, threonine residues or glycosylated residues. Glycoproteins 710may be oxidized with periodate 715 to produce a dialdehyde sugarderivative 720. The dialdehyde 720 will react spontaneously with aminelabels 725 to form a Schiff's base labeled protein 730. The Shiff's base730 reaction is reversible. Reduction with sodium borohydride 735, forexample, results in an irreversibly labeled protein 740. In anotheralternative, serine or threonine residues on a protein 745 may beoxidized, for example with galactose oxidase 750, to form an aldehydederivatized protein 755. The aldehyde derivatized protein 755 may reactwith an amine label 725 to form a Shiff's base labeled protein 760.Again, reduction with sodium borohydride 735 produces an irreversiblylabeled protein 765.

The skilled artisan will realize that the disclosed methods areexemplary only. Many methods for side-chain specific protein labelingare known in the art (e.g., Bell and Bell, Proteins and Enzymes, Ch. 7,pp. 132-183, Prentice-Hall, Inc., Englewood Cliffs, N.J., 1988) and anysuch known method may be used to label proteins with a photolabel,electrical label, or any other type of label disclosed herein orotherwise known in the art.

Example 5 Raman Detection of Analytes

Methods and Apparatus

In certain non-limiting methods, Raman spectroscopy may be used foranalysis of analytes. In a non-limiting example, the excitation beam ofa Raman detection unit was generated by a titanium:sapphire laser (Miraby Coherent) at a near-infrared wavelength (750˜950 nm) or a galliumaluminum arsenide diode laser (PI-ECL series by Process Instruments) at785 nm or 830 nm. Pulsed laser beams or continuous beams were used. Theexcitation beam was passed through a dichroic mirror (holographic notchfilter by Kaiser Optical or a dichromatic interference filter by Chromaor Omega Optical) into a collinear geometry with the collected beam. Thetransmitted beam passed through a microscope objective (Nikon LUseries), and was focused onto the Raman active substrate where targetanalytes (nucleotides or purine or pyrimidine bases) were located.

The Raman scattered light from the analytes was collected by the samemicroscope objective, and passed the dichroic mirror to the Ramandetector. The Raman detector comprised a focusing lens, a spectrograph,and an array detector. The focusing lens focused the Raman scatteredlight through the entrance slit of the spectrograph. The spectrograph(Acton Research) comprised a grating that dispersed the light by itswavelength. The dispersed light was imaged onto an array detector(back-illuminated deep-depletion CCD camera by RoperScientific). Thearray detector was connected to a controller circuit, which wasconnected to a computer for data transfer and control of the detectorfunction.

For surface-enhanced Raman spectroscopy (SERS), the Raman activesubstrate consisted of metallic nanoparticles or metal-coatednanostructures. Silver nanoparticles, ranging in size from 5 to 200 nm,was made by the method of Lee and Meisel (J. Phys. Chem., 86:3391,1982). Alternatively, samples were placed on an aluminum substrate underthe microscope objective. The Figures discussed below were collected ina stationary sample on the aluminum substrate. The number of moleculesdetected was determined by the optical collection volume of theilluminated sample. Detection sensitivity down to the single moleculelevel was demonstrated.

Single nucleotides may also be detected by SERS using a 100 μm or 200 μmmicrofluidic channel. Nucleotides may be delivered to a Raman activesubstrate through a microfluidic channel (between about 5 and 200 μmwide). Microfluidic channels may be made by molding polydimethylsiloxane(PDMS), using the technique disclosed in Anderson et al. (“Fabricationof topologically complex three-dimensional microfluidic systems in PDMSby rapid prototyping,” Anal. Chem. 72:3158-3164, 2000).

Where SERS was performed in the presence of silver nanoparticles, thenucleotide, purine or pyrimidine analyte was mixed with LiCl (90 μMfinal concentration) and nanoparticles (0.25 M final concentrationsilver atoms). SERS data were collected using room temperature analytesolutions.

Oligonucleotides prepared by rolling circle amplification were alsoanalyzed by Raman spectroscopy. One picomole (pmol) of a rolling circleamplification (RCA) primer was added to 0.1 pmol of circular,single-stranded M13 DNA template. The mixture was incubated with 1× T7polymerase 160 buffer (20 mM (millimolar) Tris-HCl, pH 7.5, 10 mM MgCl₂,1 mM dithiothreitol), 0.5 mM dNTPs and 2.5 units of T7 DNA polymerasefor 2 hours at 37° C., resulting in formation of an RCA product. Anegative control was prepared by mixing and incubating the same reagentswithout the DNA polymerase.

One μL of the RCA product and 1 μL of the negative control sample wereseparately spotted on an aluminum tray and air-dried. Each spot wasrinsed with 5 μL of 1× PBS (phosphate buffered saline). The rinse wasrepeated three times and the aluminum tray was air-dried after the finalrinse. One milliliter of silver colloid solution was diluted with 2 mLof distilled water. Eight microliters of the diluted silver colloidsolution was mixed with 2 μL of 0.5 M LiCl and added to the RCA productspot on the aluminum tray. The same solution was added to the negativecontrol spot. The Raman signals were collected as disclosed above.

Results

Nucleoside monophosphates, purine bases and pyrimidine bases wereanalyzed by SERS, using the system disclosed above. Table 1 shows thepresent detection limits for various analytes. TABLE 1 SERS Detection ofNucleoside Monophosphates, Purines and Pyrimidines Number of AnalyteFinal Concentration Molecules Detected dAMP 9 picomolar (pM) ˜1 moleculeAdenine 9 pM ˜1 molecule dGMP 90 μM 6 × 10⁶ Guanine 909 pM 60 dCMP 909μM 6 × 10⁷ Cytosine 90 nM 6 × 10³ dTMP 9 μM 6 × 10⁵ Thymine 90 nM 6 ×10³

Conditions were optimized for adenine nucleotides only. LiCl (90 μMfinal concentration) was determined to provide optimal SERS detection ofadenine nucleotides. Detection of other nucleotides may be facilitatedby use of other alkali-metal halide salts, such as NaCl, KCl, RbCl orCsCl. The claimed methods are not limited by the electrolyte solutionused, and it is contemplated that other types of electrolyte solutions,such as MgCl, CaCl, NaF, KBr, LiI, etc. may be of use. The skilledartisan will realize that electrolyte solutions that do not exhibitstrong Raman signals will provide minimal interference with SERSdetection of nucleotides. The results demonstrate that the Ramandetection system and methods disclosed above were capable of detectingand identifying single molecules of analyte

FIG. 8 shows the Raman emission spectra of a 100 mM solution of fourdifferent types of nucleotides, in the absence of surface enhancementand without Raman labels. No LiCl was added to the solution. A 10 seconddata collection time was used. Excitation occurred at 514 nm. Lowerconcentrations of nucleotides may be detected with longer collectiontimes, added electrolytes and/or surface enhancement. As shown in FIG.8, the unenhanced Raman spectra showed characteristic emission peaks foreach of the four unlabeled nucleoside monophosphates.

Surface-enhanced Raman emission spectra were obtained for a 1 nMsolution of guanine, a 100 nM solution of cytosine, and a 100 nMsolution of thymine in the presence of LiCl and silver nanoparticles(not shown). A 785 nm excitation wavelength was used. Each spectrumexhibited distinguishable Raman emission peaks (not shown).

FIG. 9 shows the SERS spectrum of a 500 nM solution of dATP (lowertrace) and fluorescein-labeled dATP (upper trace), with excitation at785 nm. dATP-fluorescein was purchased from Roche Applied Science(Indianapolis, Ind.). The Figure shows a strong increase in SERS signalupon labeling with fluorescein.

An RCA product was detectable by SERS, with emission peaks at about 833and 877 nm (not shown). Under the conditions of this protocol, with anLiCl enhancer, the signal strength from the adenine moieties wasstronger than those for guanine, cytosine and thymine. The negativecontrol (not shown) showed that the Raman signal was specific for theRCA product, as no signal was observed in the absence of amplification.

The skilled artisan will realize that the disclosed methods areexemplary only and that the Raman detection techniques disclosed foranalysis of nucleotides and oligonucleotides are also applicable foramino acids and proteins. Using the disclosed techniques, specific typesof amino acid residues on proteins may be covalently labeled, forexample with a Raman tag. The labeled protein may be passed through ananopore and the tagged residues detected, for example by Ramanspectroscopy. As disclosed above, Raman spectroscopy may be used todetected tagged or untagged residues at the single molecule level.Detection of tagged amino acid residues may be used to constructdistances maps, to identify and/or sequence proteins or other analytesof interest.

Example 6 Raman Detection of Amino Acids, Proteins and Peptides

Raman spectroscopy of amino acids, proteins and peptides was performedas disclosed in Example 5. As shown in FIG. 10 through FIG. 17, the SERSspectra of tryptophan, cysteine, methionine, histidine, phenylalanine,arginine and tyrosine all showed distinguishable SERS spectra, withcharacteristic Raman emission peaks. Derivatization of the amino acidsalso resulted in changes in the Raman spectra (compare FIG. 10 versusFIG. 17 for the SERS spectra of 1 mM tryptophan versus5-fluorotryptophan). Attachment of a fluorine residue resulted in aconsiderable change in the SERS spectrum of tryptophan (FIG. 10 and FIG.17). The spectrum was also dependent upon the position at which thefluorine residue was attached, with 5-fluorotryptophan (Sigma Chemicals,St. Louis, Mo.) giving a different SERS spectrum from 6-fluorotryptophan(not shown).

As discussed in the proceeding Example, it is concluded that Ramanspectroscopic techniques, such as SERS, may be used to identify anddistinguish different types of amino acid residues. The SERS spectrashown in FIG. 10 through FIG. 16 were obtained for unlabeled aminoacids. As shown in FIG. 9 for nucleotide residues, covalent modificationwith a Raman label may produce a large increase in signal strength forthe derivatized residue. As discussed above, side-chain specificlabeling may be used to attach different types of Raman tags todifferent types of amino acid residues.

SERS spectra were also obtained for whole proteins. FIG. 18 shows anexemplary SERS spectrum for 1% calf serum dried on an aluminum plate.Emission peaks were observed at 829, 839, 848, 852, 865, 872, 877, 886and 896 nm. The SERS spectrum of 1% bovine serum albumin was similar,with slight differences observed in the spectrum (not shown). SERSdetection of whole calf serum resulted in detection of characteristicpeaks down to 0.1% serum (FIG. 19 and FIG. 20). FIG. 19 shows the SERSspectrum of 100% whole calf serum. A 160 μl aliquot of silvernanoparticles (diluted 1:2 with distilled water) was mixed with 20 μl ofcalf serum and 40 μl of 0.5 M LiCl. A 1 second collection time resultedin the spectrum shown in FIG. 19. The SERS spectrum showed emissionpeaks at 826, 832, 839, 842, 848, 854, 862, 873, 876 and 886 nm. Asample of 0.1% calf serum still resulted in a detectable SERS spectrum(FIG. 20), with detectable peaks at 842, 848, 854, 876, 882 and 886 nm.

SERS spectra were also obtained for a series of peptides, generated bytrypsin digestion of serum proteins. The digested peptides wereseparated by reverse-phase HPLC on a C18 column. Elution of peptidesoccurred in an acidic mixture of trifluoroacetate and acetonitrile. Theresulting peptides were analyzed by SERS spectroscopy (FIG. 21). It canbe seen that different peptides showed distinguishable SERS spectra,with different sized peaks occurring at 827, 833, 844, 848, 853, 857,859, 862, 870, 874 and 880 nm.

The results obtained demonstrate that SERS spectroscopy may be used toidentify and distinguish different types of amino acid residues withinproteins or peptides, allowing the production of distance maps forproteins detection, identification and/or sequencing.

All of the METHODS and APPARATUS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. It will be apparent to those of skill in the art thatvariations may be applied to the METHODS and APPARATUS described hereinwithout departing from the concept, spirit and scope of the claimedsubject matter. More specifically, it will be apparent that certainagents that are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the claimed subject matter.

1. A method comprising: a) obtaining one or more labeled proteins,polypeptides or peptides; b) passing the labeled proteins, polypeptidesor peptides through one or more nanopores; c) detecting labeled aminoacid residues in the labeled proteins, polypeptides or peptides; d)compiling an amino acid distance map for each type of labeled aminoacid; and e) identifying the protein based on the distance maps.
 2. Themethod of claim 1, further comprising: a) placing a template nucleicacid into at least one chamber, each chamber to contain a different typeof labeled amino acid; and b) producing one or more labeled proteins,polypeptides or peptides encoded by the template nucleic acid.
 3. Themethod of claim 1, further comprising: a) obtaining one or moreproteins, polypeptides or peptides from a biological sample; and b)labeling the proteins, polypeptides or peptides post-translationally. 4.The method of claim 1, wherein the protein, polypeptide or peptide isidentified by comparing the distance maps with a library of amino aciddistance maps.
 5. The method of claim 1, wherein the protein,polypeptide or peptide is identified by comparing the distance maps withthe sequences of known proteins.
 6. The method of claim 2, wherein eachchamber is operably coupled to a different set of nanopores.
 7. Themethod of claim 1, wherein each nanopore is operably coupled to adetector.
 8. The method of claim 1, wherein only one labeled protein,polypeptide or peptide passes through a nanopore at a time.
 9. Themethod of claim 2, wherein the labeled amino acids in each chamberrepresent between about 0.5% and about 50% of the total amount of thesame amino acid in that chamber.
 10. The method of claim 1, wherein thelength of time between passage of a first labeled amino acid through thenanopore and passage of a second labeled amino acid through the nanoporecorresponds to the distance along the labeled protein, polypeptide orpeptide between the first and second amino acids.
 11. The method ofclaim 1, wherein the labels are selected from the group consisting ofluminescent labels, fluorescent labels, phosphorescent labels,chemiluminescent labels, conductive labels, nuclear magnetic resonancelabels, mass spectroscopy labels, electron spin resonance labels,electron paramagnetic resonance labels and Raman labels.
 12. The methodof claim 1, wherein at least one end of the labeled protein, polypeptideor peptide is attached to an identifiable label.
 13. The method of claim1, wherein said labeled amino acids are detected with a photodetector.14. The method of claim 1, wherein said labeled amino acids are detectedwith an electrical detector.
 15. The method of claim 2, furthercomprising analyzing a multiplicity of labeled proteins, polypeptides orpeptides from each chamber.
 16. The method of claim 1, furthercomprising determining at least a partial sequence of the protein,polypeptide or peptide based on the distance maps.
 17. An apparatuscomprising: a) at least one sub-device, each sub-device comprising anfirst chamber and a second chamber, said first and second chambersseparated by sensor layers, the first and second chambers of eachsub-device in fluid communication through one or more nanopores; and b)one or more detectors operably coupled to the nanopores.
 18. Theapparatus of claim 17, further comprising an electrode in each first andsecond chamber to provide an electrical potential gradient between thefirst and second chambers.
 19. The apparatus of claim 17, furthercomprising a computer operably coupled to the one or more detectors. 20.The apparatus of claim 17, wherein the one or more detectors comprise aphotodetector, an electrical detector and/or a voltage detector.
 21. Theapparatus of claim 20, wherein the one or more detectors comprise aRaman detector.
 22. The apparatus of claim 17, wherein said sensorlayers comprise a support layer, a photon sensing layer and two or morelight opaque layers.
 23. The apparatus of claim 22, further comprising alight source and an amplifier.
 24. The apparatus of claim 17, whereinsaid sensor layers comprise at least one conducting layer and at leasttwo insulating layers.
 25. The apparatus of claim 24, wherein saidconducting layer is operably coupled to one or more electricaldetectors.
 26. The apparatus of claim 17, wherein said nanopore is partof a nanotube or nanochannel.
 27. A method comprising: a) contacting oneor more cells with a labeled subunit; b) obtaining one or more copies ofa molecule comprising labeled subunits from the cells; c) passing thelabeled molecule through one or more nanopores; d) detecting labeledsubunits on the labeled molecule; e) compiling a subunit distance map;and f) identifying the molecule from the distance map.
 28. The method ofclaim 27, wherein the molecule is selected from the group consisting ofa nucleic acid, oligonucleotide, protein, polypeptide, peptide,polysaccharide and lipid.
 29. The method of claim 28, wherein themolecule is a protein, polypeptide or peptide and the cells aretransformed with an expression vector encoding the protein, polypeptideor peptide.
 30. The method of claim 27, further comprising contacting atleast two groups of cells with labeled subunits, each group of cellscontacted with a different type of labeled subunit.